Temperature-sensitive liposomal formulation

ABSTRACT

A liposome contains an active agent and has a gel-phase lipid bilayer membrane comprising phospholipid and a surface active agent. The phospholipids are the primary lipid source for the lipid bilayer membrane and the surface active agent is contained in the bilayer membrane in an amount sufficient to increase the percentage of active agent released at the phase transition temperature of the lipid bilayer, compared to that which would occur in the absence of the surface active agent. The surface active agent is present in the lipid bilayer membrane so as to not destabilize the membrane in the gel phase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application is a continuation-in-part application of Ser.No. 09/099,668 filed 18 Jun. 1998, the disclosure of which isincorporated herein by reference in its entirety.

This invention was made with Government support under NationalInstitutes of Health grant NIH GM40162 and National Cancer InstituteSPORE grant P50-CA68438. The Government may have certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention relates to thermosensitive liposomes, and morespecifically to liposomes comprising phospholipids and a surface activeagent, wherein the liposomes release their contents at mild hyperthermictemperatures.

BACKGROUND OF THE INVENTION

Liposomes consist of at least one lipid bilayer membrane enclosing anaqueous internal compartment. Liposomes may be characterized by membranetype and by size. Small unilamellar vesicles (SUVs) have a singlemembrane and typically range between 0.02 and 0.05 μm in diameter; largeunilamellar vesicles (LUVs) are typically larger than 0.05 μm.Oliglamellar large vesicles and multilamellar large vesicles havemultiple, usually concentric, membrane layers and are typically largerthan 0.1 μm. Liposomes with several nonconcentric membranes, i.e.,several small vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

Conventional liposomes are formulated to carry therapeutic agents, drugsor other active agents either contained within the aqueous interiorspace (water soluble active agents) or partitioned into the lipidbilayer (water-insoluble active agents). Copending U.S. patentapplication Ser. No. 08/795,100 discloses liposomes containingcholesterol in the lipid bilayer membrane, where an active agent isaggregated with a lipid surfactant to form micelles and the micelles areentrapped in the interior space of the liposome.

Active agents that have short half-lives in the bloodstream areparticularly suited to delivery via liposomes. Many anti-neoplasticagents, for example, are known to have a short half-life in thebloodstream such that their parenteral use is not feasible. Thesecompounds also believed to distribute widely to many organs and tissuesof the body to which they are toxic, thereby often limiting theconcentrations that can be injected parentally. Encapsulation withinliposomes typically helps to reduce this toxicity. Thus, the main goalsof drug delivery are to retain drug in a biocompatible capsule therebyreducing toxicity, to avoid the body's defenses that normally recognizeforeign particles and target them for removal by the liver and spleen,to instead allow targeting of the drug carrier to the therapeutic siteof action, and once there, to release the drug rapidly so that it canact on the target tumor tissue. Conventional liposomes successfullyachieve the first criterion, but, their use for site-specific deliveryof active agents via the bloodstream is often limited by the rapidclearance of liposomes from the blood by cells of thereticuloendothelial system (RES). This problem was addressed byincorporating polyethyleneglycol lipids into the liposome membrane, thatinhibits the protein adsorption that labels the liposome for RES uptake.Even if the liposomes can be made to accumulate at a diseased site suchas a solid tumor, the drug is not necessarily released and available forefficacious activity; that ability to retain the drug often becomes aninhibitory factor at the tumor site.

Liposomes are normally not leaky but will become so if a hole occurs inthe liposome membrane, if the membrane degrades or dissolves. Such abreakdown in permeability can be induced by the application of electricfields (electroporation),or exposure of the liposome to enzymes, orsurfactants. Another, method involves raising the temperature of themembrane to temperatures in the vicinity of its gel to liquidcrystalline phase transition temperature, where it appears that porousdefects at phase boundary regions in the partially liquid and partiallysolid membrane allow the increased transport of water, ions and smallmolecules through the membrane. The clinical elevation of temperature inthe body is called hyperthermia. This procedure has been used to raisethe temperature at a target site in a subject and iftemperature-sensitive liposomes can be delivered to the target site thenthis increase in temperature can cause the release of liposome contents,giving rise to the selective delivery of therapeutic agents, asinitially described by Yatvin et al., Science 204:188 (1979). Thistechnique is limited, however, where the phase transition temperature ofthe liposome is significantly higher than the normal tissue temperature.

As an example, in order to begin to use this technology for thetreatment of deep-seated tumors (e.g., prostate, ovarian, colorectal andbreast tumors), it is accordingly desirable to devise liposomeformulations capable of delivering therapeutic amounts of active agentsin response to mild hyperthermic conditions, i.e., for clinicallyattainable temperatures in the range 39-41° C.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a liposome containing an activeagent. The liposome has a solid (e.g., gel)-phase lipid bilayer membranecomprising phospholipid and a surface active agent. The phospholipidsare a primary lipid source for the lipid bilayer membrane. The surfaceactive agent is contained in the bilayer membrane in an amountsufficient to increase the percentage of active agent released at thephase transition temperature of the lipid bilayer compared to that whichwould occur in the absence of the surface active agent. The surfaceactive agent is present in the lipid bilayer membrane such that themembrane is stable in the gel-phase, i.e., the presence of the surfaceactive agent does not destabilize the membrane, particularly prior tothe melting of the lipid bilayer.

In another aspect, the invention provides a liposome containing anactive agent. The liposome has a gel-phase lipid bilayer membranecomprising phospholipid and a second component. Phospholipids are theprimary lipid source for the lipid bilayer membrane and the secondcomponent is contained in the bilayer membrane in an amount sufficientto increase the percentage of material to be released at the phasetransition temperature of the lipid bilayer compared to that which wouldoccur in the absence of the second component. The second component ispresent in the lipid bilayer membrane so as to not destabilize themembrane prior to the melting of the lipid bilayer. The material to bereleased from the liposome is the second component or a third componentwhich is entrapped within the liposome interior or associated with thelipid bilayer membrane.

In other aspects, the invention also provides methods for makingliposomes and methods of administering liposomes as described in greaterdetail herein.

These and other aspects and advantages of the invention are set forth indetail hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents a liposome having a bilayer membranecontaining dipalmitoylphosphatidylcholine (DPPC) as a phospholipid andmonopalmitoylphosphatidylcholine (MPPC) as a lysolipid. The orientationof the lysolipid monomers and their presence in both the inner and outerlayers of the lipid bilayer is indicated.

FIG. 2 graphs the effect of extrusion pass on the mean diameter of DPPCliposomes containing DPPC:MPPC (90:10). Multilamellar vesicles with anaverage size of 700 nm were extruded through a stack of twopolycarbonate membranes of pore size 0.1 mM under a pressure of 300-400psi at 45°C.

FIG. 3 graphs the release of 6-Carboxyfluorescein (CF) entrapped inliposomes composed of DPPC:MPPC (90:10), as a function of time in thepresence of PBS at 37° (open circles) and 400° C. (closed squares).

FIG. 4 graphs the release of CF from DPPC:MPPC liposomes of variedconcentrations, at temperatures between 20° C. and 45° C. in thepresence of 10 mM PBS (pH=7.4). Liposomes contained DPPC alone (opencircles); DPPC:MPPC 98:2 (closed squares); DPPC:MPPC 96:4 (closedtriangles); DPPC:MPPC 94:6 (open triangles V); DPPC:MPPC 93:7 (closeddiamonds); DPPC:MPPC 92:8 (open squares); DPPC:MPPC 90:10 (closedcircles); DPPC:MPPC 80:20 (closed triangles).

FIG. 5A provides heat flow thermograms showing the effect of varied MPPCconcentration on the phase transition temperature (Tc) of DPPCliposomes. The Tc of lyophilized liposomal samples of DPPC containingMPPC (1-10 mol %) was measured by Differential Scanning Calorimetry(DSC) between 30° C.-45° C. with 2° C./minute heating rate.

FIG. 5B graphs the effect of MPPC concentration on the phase transitiontemperature (Tc) of DPPC liposomes, as described above for FIG. 5A. Thegraph shows the start point of the transition (open circle); the peak inenthalpy (closed circle); and the end point of the transition (opentriangle).

FIG. 6A compares the differential scanning calorimetric profile (excessheat flow; open squares) of liposomes (90:10 DPPC:MPPC) with thedifferential release profile (solid circles) for 6-Carboxyfluorescein(CF) release, as obtained from the cumulative release experimentdescribed in FIG. 4.

FIG. 6B graphs the differential release profile from 90:10 DPPC:MPPCliposomes, where open circles represent heat flow and solid circlesrepresent differential release of CF over temperatures of from 25° C. to45° C.

FIG. 7 graphs the release of entrapped Doxorubicin (DX) from liposomes(90:10 DPPC:MPPC) as a function of time at 37° C. (solid circles) and40° C. (open circles) in the presence of PBS.

FIG. 8 graphs the cumulative release profiles of entrapped DX fromliposomes composed of 90:10 DPPC:MPPC, incubated at temperatures ofbetween 25° C. and 45° C. for five minutes in the presence of PBS.

FIG. 9A graphs the release of entrapped 6-Carboxyfluorescein (CF) fromliposomes (90:10 DPPC:MPPC incorporated with 5 mol % of DSPE-PEG2000) inthe presence of PBS and as a function of temperature (37° C.—opencircle; 38° C.—closed square; 39° C.—closed triangle; 39.5° C.—opentriangle; and 40° C.—closed circle).

FIG. 9B graphs the release of entrapped 6-Carboxyfluorescein (CF) fromliposomes (composed of 90:10 DPPC:MPPC and incorporated with 5 mol % ofDSPE-PEG2000) in the presence of 50% bovine serum and as a function oftemperature (37° C.—open circle; 38° C.—closed square; 39° C.—closedtriangle; 39.5° C.—open triangle; and 40° C.—closed circle).

FIG. 10 graphs the cumulative release of entrapped 6-Carboxyfluorescein(CF) from liposomes composed of 90:10 DPPC:MPPC, at various temperaturesfrom 25° C. to 45° C. in the presence of PBS (closed circles) or 50%bovine serum (open circles).

FIG. 11A shows the chemical structure of1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC).

FIG. 11B shows the chemical structure of1-Palmitoyl-2-Hydoxy-sn-Glycero-3-Phosphocholine (MPPC).

FIG. 11C shows the chemical structure of1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Poly(ethyleneglycol)2000] (DSPE-PEG2000).

FIG. 12 shows the microboundary structure of a non-ideally mixedliposome bilayer membrane of the invention.

FIG. 13 shows the transition enthalpy for lipid bilayers having variousconcentrations of DPPC and MPPC.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in reference to embodimentsset forth herein and in the figures. These embodiments are merely forthe purposes of illustration and are not to be interpreted as limitingthe invention as defined by the claims.

The present invention provides liposomes that are sensitive toalterations in the temperature of the surrounding environment. In oneaspect, the temperature-sensitivity of such liposomes allows the releaseof compounds entrapped within the interior aqueous space of theliposome, and/or the release of compounds associated with the lipidbilayer, at a target site that is either heated (as in the clinicalprocedure of hyperthermia) or that is at an intrinsically highertemperature than the rest of the body (as in inflammation). Liposomebilayers of the present invention include (in addition to a primary ormain lipid component) lysolipid, or another surface active agent(s). Theinclusion of lysolipid and/or other surface active agent in the liposomebilayer enhances the release of compounds when the liposome temperaturereaches the gel-to-liquid crystalline phase transition temperature ofthe primary lipid component. In one embodiment, the presence of thelysolipid also causes the liposome to release the drug at a slightlylower temperature than that achieved with liposomes composed solely ofphospholipids. This may also be effected by employing other surfaceactive agents. As an example, liposomes of the present invention areparticularly useful in drug delivery, where the liposome contains acompound to be delivered to a preselected target site in a subject'sbody. The target site may be either artificially heated (hyperthermia)so that it is at or above the gel-to-liquid crystalline phase transitiontemperature, or the target site may be at a higher temperature thannon-targeted sites in the body due to natural causes (e.g.,inflammation), where that temperature is at or above the gel-to-liquidcrystalline phase transition temperature of the liposome utilized.

In one embodiment, when liposomes are incubated for several minutes attemperatures in the region of the gel-to-liquid crystalline phasetransition temperature (Tc) of the primary lipid composing the liposome,the liposome bilayer becomes permeable and releases solutes entrappedwithin the liposome into the surrounding solution. The clinical use ofhyperthermia with such thermally-sensitive liposomes has been proposed.See, e.g., Yatvin et al., Science 202:1290 (1978).

U.S. Pat. No. 5,094,854 (Ogawa et al.) discloses liposomes in which theosmotic pressure of the drug-containing solution entrapped in liposomesis 1.2-2.5 times higher than that of the body fluid of warm-bloodedanimals. The temperature range in which the liposome membrane becomespermeable to material release is stated to be in the range of 40° C. to45° C.

U.S. Pat. No. 5,720,976 (Kim et al.) discloses the use of a copolymer ofN-isopropylacrylamide/octadecylacrylate/acrylic acid to coat theliposomal surface to effect the release of agents contained within theliposome. Release of the entrapped agent occurs at temperatures above28° C. (well below average human temperature).

Methods of heating a subject's body for therapeutic purposes or toassist in the delivery of therapeutic or diagnostic agents are known inthe art. Hyperthermia consists of heating diseased sites, such as solidtumors, to temperatures higher than the physiological temperature (e.g.,from about 38° C. to about 45° C.), and is currently used mainly as anadjunct to radiation therapy (Bates and Mc Killop Cancer Res. 46:5477(1986); Herman, Cancer Res. 43, 511 (1983)).

As used herein, the term “hyperthermia” refers to the elevation of thetemperature of a subject's body, or a part of a subject's body, comparedto the normal temperature of the subject. Such elevation may be theresult of a natural process (such as inflammation) or artificiallyinduced for therapeutic or diagnostic purposes. In mammals, a normalbody temperature is ordinarily maintained due to the thermoregulatorycenter in the anterior hypothalamus, which acts to balance heatproduction by body tissues with heat loss. “Hyperthermia” refers to theelevation of body temperature above the hypothalamic set point due toinsufficient heat dissipation. In contrast to hyperthermia, “fever”refers to an elevation of body temperature due to a change in thethermoregulatory center. The overall mean oral temperature for a healthyhuman aged 18-40 years is 36.8±0.4° C. (98.2±0.7° F.). See, e.g.,Harrison's Principles of Internal Medicine (Fauci et al., Eds.) 14^(th)Edition, McGraw-Hill, New York, p. 84 (1998).

The use of hyperthermia with thermally-sensitive liposomes has beenproposed, for example, for the treatment of tumors (Yatvin et al.,Science 202:1290 (1978)). Using localized hyperthermia andthermally-sensitive liposomes to target anti-neoplastic agents to tumorsites in a subject acts to decrease undesirable side effects of theagent, and to enhance therapeutic results. However, the efficacy ofliposomes targeted to diseased sites by hyperthermia depends on thestability of the liposome in the blood stream (when liposomes areadministered to the circulatory system), and on the amount of activeagent released by the liposome at the target site. For example,liposomes described by Yatvin et al, Science 202:1290 (1978) releasedonly a portion of the drug carried by the liposome at hyperthermictemperatures.

As used herein, “hyperthermic administration” of an active agent refersto its administration in conjunction with the use of clinicalhyperthermia in the subject at a preselected target site, to deliver alarger amount of active agent to the target site compared to that whichwould result from the administration of the active agent in the absenceof hyperthermia.

In one embodiment, liposomes of the present invention comprise a lipidpossessing a gel-to-liquid crystalline transition temperature in thehyperthermic range (e.g., the range of from approximately 38° C. toapproximately 45° C.). Preferred are phospholipids with aphase-transition temperature of from about 38° C. to about 45° C., andmore preferred are phospholipids whose acyl groups are saturated. Aparticularly preferred phospholipid is dipalmitoylphosphatidylcholine(DPPC). DPPC is a common saturated chain (C16) phospholipid with abilayer transition of 41.5° C. (Blume, Biochemistry 22:5436 (1983);Albon and Sturtevant, Proc. Natl. Acad. Sci. USA 75:2258 (1978)).Thermosensitive liposomes containing DPPC and other lipids that have asimilar or higher transition temperature, and that mix ideally with DPPC(such 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG)(Tc=41.5° C.) and 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC)(Tc=55.1° C.)) have been studied. Kastumi Iga et al, Intl. J.Pharmaceutics, 57:241 (1989); Bassett et al, J. Urology, 135:612 (1985);Gaber et al, Pharmacol. Res. 12:1407 (1995). Thermosensitive liposomescontaining DPPC and cholesterol have also been described. Demel and DeKruyff, Biochim. Biophys. Acta. 457:109 (1976). Other examples ofphospholipids that can be employed include di-chain phospholipids (e.g.,phosphacholines) such as, but are not limited to, a C12 saturated chainphospholipid (Tc=10° C.), a C14 saturated chain phospholipid (Tc=24°C.), a C16 saturated phospholipid (Tc=41° C.), a C18 saturatedphospholipid (Tc=55° C.), a C20 saturated phospholipid (Tc=65° C.), aC22 saturated phospholipid (Tc=70° C.), and a C24 saturated phospholipid(Tc=80° C.). Similarly, other common phospholipids that may be usedinclude, but are not limited to, phosphatdyl glycerols, inositols,ethanolamines shpyngomyelins, and gangliosides that as with thephosphatidylcholines have phase transition temperatures that vary in asimilar fashion dependent of their acyl chain length.

It should be appreciated that other membrane-forming materials can beused which are not phospholipids for the purposes of the invention.Exemplary materials which may form a solid-phase membrane include, butare not limtied to, bola lipids or bacterial lipids. Additionally, blockcopolymers comprising a water-soluble polymers (e.g., polyethyleneglycol) and a water-insoluble polymer (e.g., polypropylene oxide andpolyethylethylene) can be employed.

As used herein, the “primary lipid” in a liposome bilayer is that whichis the main lipid component of liposome bilayer material. Thus, forexample, in a liposome bilayer composed of 70 mole % phospholipid and 30mole % lysolipid, the phospholipid is the primary lipid.

Liposomes of the present invention incorporate a relatively-watersoluble surface active agent, such as, for example, a lysolipid, into abilayer composed primarily of a relatively water-insoluble molecule,such as a di-chain phospholipid (e.g., DPPC). Incorporation of thesurface active agent in the gel phase of the primary lipid componentenhances the release of contents from the resulting liposome when heatedto the gel-liquid crystalline phase transition temperature of theprimary lipid. Preferred surface active agents are lysolipids, and aparticularly preferred surface active agent ismonopalmitoylphosphatidylcholine (MPPC). Other lysolipids can also beused and include, but are not limited to, monoacylphosphatydlcholineswhere the head group can be phosphatdyl glycerols, inositols,ethanolamines, or ceramides, and the single acyl chain can be forexample C8-C22, with one or more C═C double bonds in the chain.Exemplary lysolipids include, but are not limited to,monopalmitoylphosphatidylcholine (MPPC). monolaurylphosphatidylcholine(MLPC), monomyristoylphosphatidylcholine (MMPC),monostearoylphosphatidylcholine (MSPC), and mixtures thereof.

MSPC basically encompasses C12-C18 monoacyl lysolipids. It should beappreciated that longer acyl chains can be included preferably if theheadgroup is made more water soluble to make the whole molecule watersoluble. Moreover, other suitable surface active agents may include, forexample, a dichain phospholipid having chains preferably of no greaterthan C10, glycolipids, and bile salts that are quite surface active butwill enter a bilayer without dissolving it at concentrations less thantheir CMC, which can be as high as tens of milliMolar.

Suitable surface-active agents are those that are compatible with theprimary lipid of the bilayer, and that desorb when the lipid melts tothe liquid phase. It should also be appreciated that othersurface-active agents that are not completely compatible with theprimary lipid may also be employed. Additional suitable surface-activeagents for use in phospholipid bilayers include, but are not limited to,palmitoyl alcohols, stearoyl alcohols, myristoyl surfactants, stearoylsurfactants, palmitoyl surfactants polyethylene glycol, glycerylmonopalmitate, glyceryl monooleate, ceramides, PEG-ceramides, andtherapeutic lipids. Therapeutic lipids include, for example, C-18 etherlinked lysophoshpatidylchohline. Block copolymers may be used andinclude, for example, polyethylene glycol-polyethylene oxide andpolyethylene glycol-polyethylene copolymers. It will be appreciated bythose skilled in the art that many types of surface active agents thatcan be used include, but are not limited to, cationic, anonic, andneutral surface active agents, such as, for example, fatty acids,glucosides, bile acids, and block copolymers.

In one embodiment, a preferred liposome of the present inventionincorporates a bilayer-compatible lysolipid in a phospholipid bilayer,the lysolipid present in the bilayer at a concentration sufficient toenhance the release of contents (e.g., active agent or therapeuticagent) from the liposome, compared to the release of contents that wouldbe achieved using a liposome composed of only lipid alone (i.e., withoutlysolipid). The contents may be contained within the interior of theliposome or in the liposome membrane. By “enhanced release”, it is meantthat either (a) that a greater percentage of contents is released at agiven temperature, compared to the amount of contents released at thattemperature by a liposome with a bilayer composed of phospholipid only;or (b) the contents entrapped in the interior of the liposome arereleased at a lower temperature than the temperature at which release ofcontents would occur using a lipsome with a bilayer composed ofphospholipid only. It will also be appreciated that in certainembodiments, the addition of some second components may raise thetemperature of the phase transition and thereby raise the temperature atwhich the bilayer becomes permeable above that of the phospholipidalone.

The present invention provides liposomes that release entrapped orencapsulated contents at temperatures that can be achieved in clinicalsettings using mild hyperthermia. For the purposes of illustration, inone example, the present invention provides liposomes that are highlystable at body temperature (37° C.) but that become unstable and showenhanced release of entrapped compounds at temperatures beyond about 39°C. This temperature range is a few degrees below that of many previousliposomal formulations that only showed significant release attemperatures greater than 42° C. Additionally, the present liposomalformulation's combination of lipid and compatible lysolipid provides alipid/lysolipid mixture with a slightly lower gel-to-liquid crystallinetransition temperature (of the lipid bilayer) compared to that of purelipid alone, yet the gel-to-liquid crystalline transition temperature isnot broadened by the inclusion of a lysolipid.

A preferred embodiment relates to a liposome having a bilayer composedprimarily of phospholipid, and containing lysolipid in an amount thatdecreases the gel-to-liquid crystalline phase transition temperature ofthe bilayer, compared to a bilayer composed of phospholipid alone. Aparticularly preferred liposome of the present invention comprises aDPPC as the primary phospholipid and MPPC as the lysolipid, where theratio of DPPC:MPPC is from about 99:1, 98:2, 97:3, 96:4, 95:5, 90:10, toabout 80:20, 75:25, 70:30, 65:35, 60:40, or even 51:49 (by molar ratio).These ratios may apply to other phospholipids and surface active agentsset forth herein.

An additional embodiment would include amonostearoylphosphatidylcholine, that raises the transition temperatureabove that for the pure lipid, and enhances the release of entrappedcontents. This formulation, is expected to be useful for drug deliveryapplications in dogs where the natural body temperature is approximately39° C. Similarly, an additional embodiment would include amonomyrystoylphosphatidylcholine, that lowers the transition temperaturewhilst still enhancing the release of contents compared to the puremembrane forming material alone. Such a formulation would be useful fortherapies directed in cold-blooded animals where body temperature is notnecessarily regulated to 37° C.

The present invention provides a new system for delivering active agentsin a lipid carrier, wherein active agents are released from the carrierover a narrow temperature range. In one embodiment, local heating oftarget sites to mildly hyperthermic temperatures (i.e., 39° C. to 41°C.) allows preferential delivery of the active agent to a diseased site.The present liposomes are suited for use in combination withhyperthermia to target active agents to disease sites, compared toconventional liposomes that only release active agents slowly and thatare not thermosensitive, or compared to thermosensitive liposomesfundamentally different compositions (dichain phospholipids andcholesterol, that do not contain, for example, a second water-solublesurfactant) and that do not release active agents until reachingtemperatures of 42° C. or above.

While not wishing to be held to any single theory of action, the presentinventors believe that the mechanism whereby lysolipids (or othersurface active agents or active agents that may also be surface active)enhance the release of contents from liposomes in one embodimentcomposed primarily of phospholipid is related to the way in which thelysolipid is mostly ideally mixed in the mixed gel phase bilayer, butcreates defects at the microstructural level (microdomain boundaries) asit desorbs from the membrane upon bilayer melting at the primary acylchain transition temperature (i.e., at the transition temperature of theprimary bilayer lipid/surface active agent (e.g., lysolipid) mixture).The inclusion of a surface active agent such as a lysolipid lowers thephase transition temperature of a lipid bilayer membrane, compared tothe phase transition temperature of a membrane composed solely of thephospholipid. In a liposome composed of DPPC and MPPC, the phasetransition temperature is lowered depending on the amount of MPPCincorporated into the gel phase bilayer; the reduction of phasetransition temperature that can be achieved is limited by the amount ofMPPC that can be stably contained in the bilayer. Membranes composed ofphospholipid (e.g., DPPC) can stably contain from 1 mol % surface activeagent (e.g., MPPC), up to about 20 mole %, 30% mole %, 40 mol %, or even50 mol % surface active agent, depending on other conditions such as theactive agent contained within the liposome.

Additionally, surface active agent and phospholipid may be present in anon-ideally mixed form in the lipid bilayer. FIG. 12 is a diagramillustrating the phases in such a system. A non-ideally mixed systemcontains second phase precipitates that form microdomain inter-grainboundaries 10 similar to those which are in an ideally mixed systemdescribed above as well as intra-grain boundaries 20 segregating anintra-grain region (e.g., precipitated second phase) within a givenprimary phase 30. The formation of and the structure of the lipidbilayer illustrated in this embodiment may be influenced by variousfactors such as, but not limited to, types and concentrations of bilayercomponents, cooling rates, and others. Not wishing to be bound bytheory, the present inventors believe that the mechanism whereby surfaceactive agents may enhance the release of contents from liposomes in thisembodiment is related to the formation of defects at the microstructurallevel (microdomain boundaries) as it desorbs from the membrane uponbilayer melting at the primary acyl chain transition temperature (i.e.,at the transition temperature of the primary bilayer lipid/surfaceactive agent mixture). In the embodiment set forth in FIG. 12, thedefects occur at the microdomain inter-grain boundaries 10 and well asthe intra-grain boundaries 20. Thus, the enhanced release benefits canbe realized in lipid bilayers in which phospholipid(s) and surfaceactive agent are non-ideally mixed as well as ideally mixed.

In liposome bilayers containing phospholipid and a surface active agentsuch as a lysolipid, it is preferable that the surface active agent becontained in both layers of the bilayer. This concept of this embodimentis illustrated in FIG. 1, which schematically represents a liposomecomposed of DPPC and MPPC. The molecules of MPPC are present in both theexterior and the interior layer of the liposome membrane bilayer. In aliposome containing surface active agent in only one layer of thebilayer, redistribution of the surface active agent to both layers ofthe bilayer will occur over time at temperatures above the geltransition temperature (e.g., in the liquid crystalline phase).

Phase compatibility of the two (or more) components of the presentinvention affects the processing and stability of the lipid bilayerstructure. For example, in liposomes composed primarily of DPPC (adi-chain phospholipid), to maximize compatibility and preserve thenarrow melting range of the main phospholipid, a preferred surfaceactive agent is lysolipid such as MPPC because it is identical to thedi-chain phospholipid except that it possesses only one acyl chain(FIGS. 11A and 11B). In one embodiment, the present inventors discoveredthat inclusion of this bilayer-compatible lysolipid in lowconcentrations (preferably 2-20 mol %), makes liposomes composedprimarily of DPPC more “leaky” at the point at which the primary lipidbegins to melt (i.e., the solidus line of the main phase transition),compared to liposomes composed of DPPC alone. While not wishing to beheld to a single explanation, the present inventors believe that gelphase bilayers are composed of microcrystalline domains; as thetemperature approaches the gel-to-liquid crystalline phase transition ofthe lipid bilayer, membrane permeability to the entrapped drug increasesat the grain boundaries of the microstructure. At the transitiontemperature, desorption of the lysolipid dissolved in the gel phasemicrostructure enhances the membrane permeability. An additional benefitof incorporating a compatible molecule in the liposome bilayer is thatthe phase transition temperature of the primary lipid is not broadened,but is lowered (or raised depending on the application) by about adegree or more (depending on the lysolipid concentration in thebilayer).

In another embodiment, the invention provides a liposome having agel-phase lipid bilayer membrane comprising phospholipid and a secondcomponent. The phospholipids are the primary lipid source for the lipidbilayer membrane. The second component is one which is capable ofincreasing the percentage of material to be released at the phasetransition temperature compared to that which would occur in the absenceof the second component. Thus, the second component is present in thelipid bilayer membrane in an amount such that it allows for thisenhanced release. Additionally, the second component is present in thelipid bilayer membrane so as to not destabilize the membrane prior tothe melting of the lipid bilayer, i.e., the bilayer membrane is stablein the gel-phase with surface active agent being contained therein. Thematerial to be released may be the second component, or a thirdcomponent which is entrapped within the liposome interior or associatedwith the lipid bilayer membrane. The third component may encompassactive agents such as, but not limited to, those described herein.

The second component as set forth above encompasses a wide range ofsubstances. In a preferred embodiment, the second component is anamphiphilic material including, but not limited to, surface activeagents set forth herein. Materials that are released includes compoundsdescribed herein such as, for example, active agents (e.g.,pharmaceutically active agents) entrapped within the interior aqueousspace of the liposome, and/or associated with the lipid bilayer. Incertain embodiments, the second component and the material that arereleased may be the same, i.e., the second component is entrapped withinthe lipid bilayer. In this instance, an example of a second componentmay include, but not be limited to, water-insoluble or membrane-solublepharmaceutically active agents such as apoptotic agents (e.g.,ceramides) as well as platelet activating factor. Other agents mayinclude, common surfactants as listed above, and drugs themselves withlimited water solubility that can preferentially associate with a liquidlipid membrane and that can then be trapped in the membrane when themain membrane forming lipid is cooled into the gel phase. Upon raisingthe temperature to the transition region, this second component that mayitself be a drug is released from the membrane into the surroundingaqueous fluid. If this fluid is the bathing fluid in a tumor tissue orcell interior, then the active agent may be available for therapeuticaction.

In the event that the second component is the material whose released isenhanced are the same, the liposome preferably comprises from about 1 toabout 50 mol percent of the second component and more preferably fromabout 1 to about 30 mol percent of the second component.

Although not intending to be bound by theory, in the above-describedembodiments, Applicants believe that the second component may accumulateat the micrograin boundaries in the gel-phase lipid bilayer. As thebilayer melts, the second component releases or desorbs from the bilayerat these grain boundaries and thus exits the lipid bilayer in a mannerdescribed herein. Lipid bilayers that may be employed in theseembodiments include systems in which the phospholipid and secondcomponent are ideally mixed or non-ideally mixed as described above.

The process of forming the mixed component liposomes of the presentinvention involves preparation of gel phase liposomes containing aphospholipid, an appropriate surface active agent (such as lysolipid),and the active agent of interest. Other phase compatible components suchas DSPE-PEG can optionally be included, as discussed further below. Thecomposition contains a percentage of lysolipid such that the surfaceactive agent does not destabilize the membrane at processingtemperatures where the bilayer is in the liquid phase, nor atphysiological temperatures where the bilayer is in the gel phase. Thebilayer becomes unstable and permeable at temperatures in the range ofthe membrane phase transition, which can be made to be just above normalhuman body temperature, and rapidly releases entrapped material from theliposome interior.

Liposomes according to the present invention may be prepared by any of avariety of techniques that are known in the art. See, e.g., U.S. Pat.No. 4,235,871; Published PCT applications WO 96/14057; New RRC,Liposomes: A practical approach, IRL Press, Oxford (1990), pages 33-104;Lasic DD, Lipsomes from physics to applications, Elsevier SciencePublishers, Amsterdam, 1993; Lipsomes, Marcel Dekker, Inc., New York(1983). Entrapment of an active agent within liposomes of the presentinvention may also be carried out using any conventional method in theart. In preparing liposome compositions of the present invention,stabilizers such as antioxidants and other additives may be used as longas they do not interfere with the purpose of the invention.

The amount of surface active agent (such as, for example, lysolipid)included in liposomes of the present invention is not sufficient todestabilize the membrane in the liquid phase that occurs duringprocessing of the liposomes (prior to cooling to produce the gel phaseproduct). In producing liposomes according to the present invention, itis preferable during processing of the liquid phase membranes tomaintain a concentration of surface active agent monomer both inside andoutside the liposome, to avoid a concentration gradient that woulddeplete the surface active agent concentration in the membrane. This canbe achieved by preparing the liposomal suspension from a premixedmixture in an aqueous medium containing sufficient amount of surfaceactive agent in monomeric form, (e.g., at approximately but not greatlyexceeding the critical micelle concentration (CMC) of the surface activeagent).

A method of preparing a liposomal formulation according to the presentinvention comprises mixing the bilayer components in the appropriateproportions in a suitable organic solvent, as is known in the art. Thesolvent is then evaporated to form a dried lipid film. The film isrehydrated (at temperatures above the phase transition temperature ofthe lipid mixture) using an aqueous solution containing an equilibratingamount of the surface active agent and a desired active agent. Theliposomes formed after rehydration can be extruded to form liposomes ofa desired size, as is known in the art. For example, where liposomescomposed of 80:20 DPPC:MPPC are produced, rehydration is carried out ata temperature above the phase transition temperature of this particularlipid mixture (above 39° C). The aqueous solution used to rehydrate thelipid film comprises an equilibrating amount of lysolipid monomers(e.g., a concentration equal to the Critical Micelle Concentration ofMPPC, about 1 micromolar).

Conventional liposomes suffer from a relatively short half life in theblood circulation due to their rapid uptake by macrophages of the liverand spleen (organs of the reticuloendothelial system or RES), andtherefore do not accumulate in leaky tumor tissue. Liposome preparationshave been devised which avoid rapid RES uptake and which have increasedcirculation times. STEALTH® liposomes (Sequus Inc., Menlo Park Calif.)include polyethyleneglycol (PEG)-grafted lipids at about 5 mol % in thelipid bilayer. See, e.g., Allen, UCLA Symposium on Molecular andCellular Biology, 89:405 (1989); Allen et al., Biochim. Biophys. Acta1066:29 (1991); Klibanov et al., FEBS Letters 268:235 (1990); Needham etal., Biochim. Biophys. Acta 1108:40 (1992); Papahadjopoulos et al.,Proc. Natl. Acad Sci. USA 88:11460 (1991); Wu et al., Cancer Research53:3765 (1993); Klibanov and Huang, J. Liposome Research 2:321 (1992);Lasic and Martin, Stealth Liposomes, In: Pharmacology and Toxicology,CRC Press, Boca Raton, Fla. (1995). See also U.S. Pat. No. 5,225,212 toMartin et al.; U.S. Pat. No. 5,395,619 to Zalipsky et al. regardingliposomes containing polymer grafted lipids in the vesicle membrane. Thepresence of polymers on the exterior liposome surface decreases theuptake of liposomes by the organs of the RES.

Liposomes of the present invention may be formulated to includepolymer-grafted lipids, as is known in the art, to decrease liposomeuptake by the RES and thus increase the circulation time of theliposomes. Suitable polymers include hydrophilic polymers such aspolyethylene glycol, polyvinylpyrolidine, olylactic acid, polyglycolicacid, copolymers of polylactic acid and polyglycolic acid, polyvinylalcohols, polyvinylpyrrolidone, dextrans, oligosaccharides, along withmixtures of the above. It is believed that most current liposome drugdelivery systems are composed of lipids that form liquid-phase bilayersat room or body temperatures. If one of the component lipids actuallyhas a relatively high transition temperature, the liposomes areconventionally formed reproducibly at temperatures above thistransition. It is also usual for loading of active agents to be carriedout at temperatures above the phase transition of the membrane lipids,i.e., in the liquid phase of the lipid.

In view of the preceding paragraph, in another aspect the inventionprovides a method for loading active agents into liposomes. The methodcomprises providing a liposome comprising a gel-phase lipid bilayer,with the lipid bilayer comprising phospholipid. The lipid bilayer ispresent at a temperature below its phase transition temperature. Thelipid bilayer is then exposed to an active agent such that the activeagent passes into and through the lipid bilayer, entering the liposomeinterior. The method of loading active agents into liposomes allows foran increase in the percentage of active agent released at the phasetransition temperature of the liposome membrane, compared to that whichwould occur in liposomes produced by another method.

The method described above may further comprise other steps. Forexample, the method may comprise the step of cooling the liposome to atemperature below the phase transition temperature of the lipid bilayerprior to exposing the active agent to the bilayer.

In a preferred embodiment, the liposome is present in a surroundingliquid medium, and wherein the pH of the surrounding liquid medium isgreater than the pH of the interior of the liposome. This pH gradient isbelieved to facilitate loading of the active agent into the interior ofthe liposome.

Active Agents

As used herein, an active agent ‘in the interior’ or ‘entrapped within’the liposome is that which contained in the interior space of theliposome, compared to that partitioned into the lipid bilayer andcontained within the vesicle membrane itself. As used herein, an activeagent ‘within’ or ‘entrapped within’ the lipid bilayer of a liposome iscarried as a part of the lipid bilayer, as opposed to being contained inthe interior space of the liposome. Active agents may be in any formsuitable for use in liposomes, as is known in the art, including but notlimited to aqueous solutions of active agents. Aqueous solutions ofactive agents within liposomes of the present invention may be at thesame osmotic pressure as that of the body fluid of the intended subject,or at an increased osmotic pressure (see U.S. Pat. No. 5,094,854); theaqueous solutions may also contain some precipitated active agent, as isknown in the art. A preferred active agent for encapsulation in theinterior of the liposome is any water soluble, weak base agent.

The incorporation of certain active agents (such as some anesthetics) inliposomes of the present invention may additionally alter (enhance orinhibit) the release of contents from the liposome, or alter thetransition temperature of the liposome, compared to that which would beseen in a similar liposome that did not contain the active agent.

The administration of antineoplastic or antitumor drugs such asdoxorubicin, cisplatin and methotrexate using thermosensitive liposomesin combination with hyperthernia at the desired target site has beenreported. See, e.g., Magin and Weinstein In: Liposome Technology, Vol.3, (Gregoriadis, G., ed.) p. 137, CRC Press, Boca Raton, Fla. (1993);Gaber et al., Intl. J. Radiation Oncology, Biol. Physics, 36(5):1177(1996).

Active agents suitable for use in the present invention includetherapeutic drugs and pharmacologically active agents, nutritionalmolecules, cosmetic agents, diagnostic agents and contrast agents forimaging. As used herein, active agent includes pharmacologicallyacceptable salts of active agents. Suitable therapeutic agents include,for example, antineoplastics, antitumor agents, antibiotics,antifungals, anti-inflammatory agents, immunosuppressive agents,anti-infective agents, antivirals, anthelminthic, and antiparasiticcompounds. Methods of preparing lipophilic drug derivatives which aresuitable for liposome formulation are known in the art (see e.g., U.S.Pat. No. 5,534,499 to Ansell, describing covalent attachment oftherapeutic agents to a fatty acid chain of a phospholipid).

In treating tumors or neoplastic growths, suitable compounds may includeanthracycline antibiotics (such as doxorubicin, daunorubicin,carinomycin, N-acetyladriamycin, rubidazone, 5-imidodaunomycin, N30acetyldaunomycin, and epirubicin) and plant alkaloids (such asvincristine, vinblastine, etoposide, ellipticine and camptothecin).Other suitable agents include paclitaxel (TAXOL®; a diterpenes isolatedfrom the bark of the yew tree and representative of a new class oftherapeutic agents having a taxane ring structure) and docetaxol(taxotere); mitotane, cisplatin, and phenesterine.

Anti-inflammatory therapeutic agents suitable for use in the presentinvention include steroids and non-steroidal anti-inflammatorycompounds, such as prednisone, methyl-prednisolone, paramethazone,11-fludrocortisol, triamciniolone, betamethasone and dexamethasone,ibuprofen, piroxicam, beclomethasone; methotrexate, azaribine,etretinate, anthralin, psoralins; salicylates such as aspirin; andimmunosuppresant agents such as cyclosporine. Anti-inflammatorycorticosteroids and the anti-inflammatory and immunosuppressive agentcyclosporine are both highly lipophilic and are suited for use in thepresent invention. Antineoplastic agents can also be used such as, forexample, Navalbene.

Additional pharmacological agents suitable for use in liposomes of thepresent invention include anesthetics (such as methoxyflurane,isoflurane, enflurane, halothane, benzocaine, lidocane, bupivocane, andropivicane); antiulceratives (such as cimetidine); antiseizuremedications such as barbituates; azothioprine (an immunosuppressant andantirheumatic agent); and muscle relaxants (such as dantrolene anddiazepam).

Imaging agents suitable for use in the present liposome preparationsinclude ultrasound contrast agents, radiocontrast agents (such asradioisotopes or compounds containing radioisotopes, includingiodo-octanes, halocarbons, and renograf in), or magnetic contrast agents(such as paramagnetic compounds).

Nutritional agents suitable for incorporation into liposomes of thepresent invention include flavoring compounds (e.g., citral, xylitol),amino acids, sugars, proteins, carbohydrates, vitamins and fat.Combinations of nutritional agents are also suitable.

The above active agents may be used in the various liposome embodimentsdescribed, but not limited to, those described hereinabove.Additionally, it should be emphasized that the liposomes may comprise asingle pharmacologically active agent (e.g., at least one active agent)or multiple active agents, depending on the intentions of theadministrator. Embodiments utilizing multiple active agents within thesame liposome or in two separate liposome formulations administeredtogether, may provide enhanced efficacy due to syngergistic behavior bythe agents. By formulating and delivering multiple (e.g., two) activeagents in a liposome or liposomes, and using mild hyperthermia as setforth herein, the active agents could be made to accumulate and releasedto a tumor at the same time or within a similar time frame.

Administration and Liposome Size

Liposomes of the present invention may be administered using methodsthat are known to those skilled in the art, including but not limited todelivery into the bloodstream of a subject or subcutaneous orintramuscular, or intracavity (peritneum or joint, or eye etc)administration of liposomes. Where liposomes according to the presentinvention are used in conjunction with hyperthermia, the liposomes maybe administered by any suitable means that results in delivery of theliposomes to the treatment site. For example, liposomes may beadministered intravenously and thereby brought to the site of a tumor bythe normal blood flow; heating of this site can result in greaterliposome extravasation from the blood stream because of the effect ofhyperthermia on blood vasculature and moreover, once extravasated intothe tumor tissue results in the liposomal membranes being heated to thephase transition temperature so that the liposomal contents arepreferentially released at the site of the tumor.

Where treatment of a tumor or neoplasm is desired, effective delivery ofa liposome-encapsulated active agent via the bloodstream requires thatthe liposome be able to penetrate the continuous (but “leaky”)endothelial layer and underlying basement membrane surrounding thevessels supplying blood to a tumor. Liposomes of smaller sizes have beenfound to be more effective at extravasation into tumors through theendothelial cell barrier and underlying basement membrane whichseparates a capillary from tumor cells. See, e.g., U.S. Pat. No.5,213,804 to Martin et al.

As used herein, “solid tumors” are those growing in an anatomical siteother than the bloodstream (in contrast to blood-borne tumors such asleukemias). Solid tumors require the formation of small blood vesselsand capillaries to nourish the growing tumor tissue.

In accordance with the present invention, the anti-tumor oranti-neoplastic agent of choice is entrapped within a liposome accordingto the present invention; the liposomes are formulated to be of a sizeknown to penetrate the endothelial and basement membrane barriers. Theresulting liposomal formulation can be administered parenterally to asubject in need of such treatment, preferably by intravenousadministration, but also by, for example, direct injection. Tumorscharacterized by an acute increase in permeability of the vasculature inthe region of tumor growth are particularly suited for treatment by thepresent methods. Administration of liposomes is followed by heating ofthe treatment site to a temperature that results in release of theliposomal contents.

Where site-specific treatment of inflammation is desired, effectiveliposome delivery of an active agent requires that the liposome have along blood halflife, and be capable of penetrating the continuousendothelial cell layer and underlying basement membrane surroundingblood vessels adjacent to the site of inflammation. Liposomes of smallersizes have been found to be more effective at extravasation through theendothelial cell barrier and into associated inflamed regions. See,e.g., U.S. Pat. No. 5,356,633 to Woodle et al. In accordance with thepresent invention, the anti-inflammatory agent of choice is entrappedwithin a liposome according to the present invention; the liposomes areformulated to be of a size known to penetrate the endothelial andbasement membrane barriers. The resulting liposomal formulation can beadministered parenterally to a subject in need of such treatment,preferably by intravenous administration. Inflamed regions characterizedby an acute increase in permeability of the vasculature in the region ofinflammation, and by a localized increase in temperature, areparticularly suited for treatment by the present methods.

It will further be appreciated that the liposomes of the presentinvention may be utilized to deliver anti-infective agents to sites ofinfection, via the bloodstream. The use of liposomes containing avesicle-forming lipid derivatized with a hydrophilic polymer, and havingsizes ranging between 0.07 and 0.2 microns, to deliver therapeuticagents to sites of infection is described in published PCT patentapplication WO 93/19738. In accordance with the present invention, theanti-infective agent of choice is entrapped within a liposome having amembrane according to the present invention, and the resulting liposomalformulation is administered parenterally to a subject, preferably byintravenous administration. If desired, localized hyperthermia may beinduced at the site of infection to cause the preferential release ofliposomal contents at that site.

The size of liposomes in a preparation may depend upon the active agentcontained therein and/or the intended target. Liposomes of between 0.05to 0.3 microns in diameter, have been reported as suitable for tumoradministration (U.S. Pat. No. 5,527,528 to Allen et al.). Sizing ofliposomes according to the present invention may be carried outaccording to methods known in the art, and taking into account theactive agent contained therein and the effects desired (see, e.g., U.S.Pat. No. 5,225,212 to Martin et al; U.S. Pat. No. 5,527,528 to Allen etal., the disclosures of which are incorporated herein by reference intheir entirety). A preferred embodiment of the present invention is aliposome of less than 10 microns in diameter, or a liposome preparationcontaining a plurality liposomes of less than 10 microns in diameter. Ina further preferred embodiment of the present invention, liposomes arefrom about 0.05 microns or about 0.1 microns in diameter, to about 0.3microns or about 0.4 microns in diameter. Liposome preparations maycontain liposomes of different sizes. Advantageously, these liposomescomprise lipid mixtures set forth herein and are thereforetemperature-sensitive, with an ability to release contained drug, asdescribed.

In another preferred embodiment of the present invention, liposomes arefrom about 50 nm, 100 nm, 120 nm, 130 nm, 140 nm or 150 nm, up to about175 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm or 500 nm indiameter.

In one aspect of the present invention, the liposomes are prepared tohave substantially homogeneous sizes in a selected size range. Oneeffective sizing method involves extruding an aqueous suspension of theliposomes through a series of polycarbonate membranes having a selecteduniform pore size; the pore size of the membrane will correspond roughlywith the largest sizes of liposomes produced by extrusion through thatmembrane. See e.g., U.S. Pat. No. 4,737,323.

In a further aspect of the present invention, liposomes are dispersed inphysiological saline or PBS to provide an aqueous preparation ofliposomes. The aqueous preparation may further include an equilibratingamount of the surface active agent contained in the liposome bilayer, toreduce or prevent loss of the surface active agent from the liposomebilayer into solution. Liposomes composed of DPPC:MPPC may be containedin physiological saline or PBS that contains from about 1 microMolar toabout 5 microMolar of MPPC monomer.

The amount of active agent to be entrapped within or carried byliposomes according to the present invention will vary depending on thetherapeutic dose and the unit dose of the active agent, as will beapparent to one skilled in the art. In general, however, the preparationof liposomes of the present invention is designed so the largest amountof active agent possible is carried by the liposome. Liposomes of thepresent invention may be of any type, however, LUVs are particularlypreferred.

The liposomes of the invention can be used in other applications suchas, for example, an anesthetic release. Moreover, in addition to theabove, the liposomes may be used to treat various non-malignant diseasessuch as, but not limited to, psoriasis and arthritis. The administrationof the liposomes in these applications may be carried out according, butnot limited to, techniques described herein. In various embodiments,especially for anticancer therapies or where drugs act at particularpoints in the cell cycle, the liposomes may be delivered in multipleshort pulses over extended time periods, such as by employing pulseheat. Examples of active agents that could be delivered in this mannerinclude, but are not limited to, cell cycle dependent drugs such as, forexample, camptothecins and vinca alkaloids. In other embodiments, activeagents may be delivered in a single protracted release including, forexample, anthracyclines (e.g., doxorubicin). The selection of activeagents for use in the various techniques may be made by the skilledartisan.

Assessing Release of Liposome Contents

Characterization of thermosensitive liposomes by the release of anentrapped fluorescent probe, 6-Carboxyfluorescein (CF), was reported inMerlin, Eur. J. Cancer 27(8): 1031 (1979). CF was entrapped intoliposomes at a quenching concentration (50 mM); no fluorescence wasobserved for CF entrapped in the liposome. Intense fluorescence,however, developed upon release of the probe from liposomes due todilution of the CF in the suspension. The amount of the probe releasedfrom the liposomes at various temperatures could thus be quantifiedbased on fluorescence. Merlin, Eur. J Cancer., 27(8):1031 (1991),studied thermally sensitive liposomes encapsulating Doxorubicin (DX),and incorporated pegylated lipids in the bilayer to increase theircirculation time in the blood stream compared to conventionalthermosensitive liposomes. See also Maruyama et al., Biochem. Biophys.Acta, 1149:17 (1993)).

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLE 1 Preparation of Temperature—Sensitive Liposomes

DPPC liposomes containing varied molar concentrations of the lysolipidMonopalmitoylphosphatidylcholine (MPPC) were prepared and characterized.The aqueous fluorescent probe 6-Carboxyfluorescein (CF) was entrappedwithin the liposomes to act as a marker for membrane permeabilitychange; CF was incorporated into the liposomes using methods known inthe art (See, e.g., Lipsomes: A Practical Approach (1990), Editor: R. R.C. New, IRL Press, Oxford, N.Y.). The liposome components were dissolvedin chloroform (a suitable organic solvent), and the solvent wasevaporated under vacuum at 45° C. using a rotavapor to form a uniformthin film of lipids on the inner walls of a round bottom flask. Thelipid film was further dried under vacuum for 25 hours to ensurecomplete removal of traces of chloroform.

The lipid film was hydrated at 45° C. with an aqueous solution of 10 mMPBS (pH=7.4) containing 50 mM CF and 1 microM MPPC. For efficienthydration, a TEFLON® bead was used to gently etch the lipids in thepresence of aqueous medium to form a cloudy suspension of multilamellarvesicles (MLVs). The MLVs thus formed had an average size of 700 nm andwere extruded through a stack of two polycarbonate membrane filters of0.1 micrometers under 300-400 psi pressure at 45° C. (i.e., above thegel-liquid crystalline temperature of the lipid or lipid mixture) asdescribed in the method developed by Hope et al. (Biochem. Biophys. Acta812:55-65 (1985)) to obtain Large Unilamellar Vesicles by ExtrusionTechnique (LUVETs) of the desired 140 nm size. The mean diameter of thevesicles was measured by Photon Correlation Spectrometer (PCS, Coulter,N4 plus) after each extrusion pass.

As shown in FIG. 2, the size of liposomes decreased with successivepasses through the membrane and reached the minimum size after threepasses.

EXAMPLE 2 Release of Liposomal Contents

The in vitro stability and thermosensitivity of the liposomalformulations prepared as described in Example 1 and containing variousmolar fractions of MPPC was assessed by measuring the percent release ofentrapped water soluble fluorescent molecule, CF, from the aqueousinterior of the vesicle to the surrounding solution as a function ofincubation temperature (25-45° C.) in the presence of PBS. Thefluorescence of CF entrapped in the liposomes was self quenched due toits high concentration, but upon release from the liposomes and dilutioninto the suspending medium, CF developed an intense fluorescence. Afterincubation, the fluorescence intensity of the samples was measured atexcitation wavelength (λex)=470 nm and emission wavelength (λem)=520 nmafter suitable dilutions to determine the amount of CF released from theliposomes. The relative percent fluorescence intensity due to incubationat a particular temperature was calculated by comparison with the totalrelease of entrapped material obtained after disruption of the liposomesamples by adding 10% Triton X-100.

This experiment was carried out for liposomes composed of pure DPPC andfor liposomes composed of DPPC:MPPC mixtures containing 2, 4, 6, 7, 8,10 or 20 mol % MPPC, over the temperature range 20° C. to 45° C.

The amount of entrapped CF released from liposomes of the presentinvention was measured as a function of time at physiological (37° C.)and hyperthermic (40°0 C.) temperatures. Liposomes were incubated in PBSfor various time intervals and the release of CF was measuredfluorimetrically at λex=470 nm and λem=520 nm. Typical heating runs areshown in FIG. 3, for the 90:10 DPPC:MPPC composition. At 37° C. thepercent release of CF was negligible. However, on incubation at 40° C.,about 60% of the entrapped CF was released within five minutes;additional contents release only increased slightly upon furtherincubation at this temperature. Thus at a given temperature, most of thecontents are released within the first five to ten minutes of heating.

FIG. 4 shows the release of CF from liposomes incubated for five minutesat temperatures of 20° C. to 45° C. in the presence of 10 mM PBS(pH=7.4). Release of CF was measured fluorimetrically at λex=470 nm andλem=520 nm. The percent of CF released was calculated by comparing thevalues obtained with those obtained after the total release of CF(achieved by the addition of Triton X-100 to the liposome sample todissolve the liposomes and release all entrapped CF). Pure DPPCliposomes were stable up to 39.5° C. but became permeable near thetransition temperature of the phospholipid, thus causing release of someof the CF. The amount of CF released from the pure DPPC liposome was,however, only about 20% of total contents. In contrast, with increasingconcentrations of MPPC in the DPPC bilayers, liposomes showed anincreasing release of CF, with maximum release occurring for theliposomes having bilayers containing 10 mol % and 20 mol % MPPC.

These results demonstrate that the incorporation of as little as 10 mol% of MPPC into the membranes of DPPC liposomes increases the amount ofCF released by a factor of 4 (compared to the release that is seen forliposomes of DPPC alone), allowing release of up to 90% of the liposomalcontents.

The temperature release profiles also show an additional benefit ofincorporating MPPC into DPPC liposome membranes, in that the onsettemperature for release is shifted to slightly lower temperatures,starting at approximately 38° C. for liposomes containing 10 mol % and20 mol % MPPC. The release profile is sharp for these liposomes, and themaximum amount of CF is released after a rise in temperature of only adegree or so, i.e., at between 38.5° C. and 40° C. for the 10% MPPCliposomes.

These experiments demonstrate that the inclusion of MPPC in liposomebilayers made of DPPC increases the amount of contents released from theinterior of the liposome, and shifts the temperature range over whichrelease occurs into the range of 38.5° C.-40° C., which is the mildhyperthermic range.

In DPPC liposomes containing MPPC concentrations of more than 20 mol %,the liposomes became intrinsically unstable and therefore unable toretain entrapped material at temperatures above the lipid phasetransition (i.e., in the liquid phase of the lipid bilayer that occursduring processing of liposomes). Such high concentrations of MPPC canalso destabilize the gel phase bilayers at temperatures below thetransition temperature. As demonstrated previously, the mechanicalstrength of membranes decreases as more and more MPPC is included(Needham et al., Biophys. J. 73:2615 (997)), and the bilayers eventuallymake a transition to a pure micelle suspension as the mole ratio of MPPCto phospholipid goes beyond 50 mol % (Zhelev et al., Biophys. J. (inpress; 1998)). One preferred molar ratio for thermally sensitiveliposomes is 90:10, DPPC:MPPC.

EXAMPLE 3 Phase Transition Behavior of DPPC/MPPC Mixtures andCorrelations with Release Temperatures

To investigate the biophysical mechanism involved in the permeability ofthe liposomes of the present invention, differential scanningcalorimetric (DSC) studies were carried out to generate differentialcalorimetric thermograms for the present liposomes, to determine thephase transition temperature. These results were compared with therelease versus temperature scans obtained from cumulative releaseprofiles (shown in FIG. 4). FIGS. 5A and 5B show the heat flowthermograms for liposome preparations containing increasingconcentrations of MPPC in DPPC bilayers. These thermograms show that thetransition temperature remains unbroadened even though up to 10% of MPPCare included in the bilayer. At a higher level of resolution, FIG. 5Bshows the change in the peak of the transition temperature from 41.9° C.to 41.04° C. as the MPPC composition is increased from zero to 10 mol %in DPPC bilayers. Also shown is the breadth of the transition,represented as the start and end point of the transition, i.e., thesolidus and liquidus lines below and above the excess heat flow peak.

The differential scanning thermogram of liposomes of the presentinvention can be compared with the differential release profiles. FIG.6A shows the cumulative release profile for CF release from theDPPC:MPPC 90:10 liposomes versus temperature, and the heat flowthermogram over the same temperature range. FIG. 6B shows thedifferential release, which highlights the sharpness of the releaseprofile and the temperature at which maximum release occurs in relationto the heat flow thermogram. What is striking about this comparison isthat that the peak release of contents obtained from the differentialrelease profiles was 0.9° C. lower than the peak in the transitionenthalpy obtained by DSC. The release of the entrapped material at thetemperature prior to Tc can be attributed to the fact that the releaseis occurring at the ‘solidus’ line of the thermogram and not at the peaktemperature. One explanation for such a behavior is that the release ofentrapped material occurs as soon as the ‘first defects’ (meltingdefects) in the microdomain boundaries of the bilayer network appear; itis here that the lysolipid may exert its effects. While not wishing tobe held to a single theory, the present inventors suspect that as thetransition temperature is approached the first parts of themicrostructure that melt are at the grain boundaries of the solidmembrane. When surrounded by a lysolipid-free aqueous phase, thelysolipid is trapped in the gel phase but can desorb when the membranebegins to melt; as it does so it enhances the defect permeability andthe contents are released more effectively than in liposomes of purelipid alone.

EXAMPLE 4 Entrapment and Release of Doxorubicin

Doxorubicin (DX) was entrapped into the inner aqueous volumes ofliposomes of the present invention (DPPC:MPPC 90:10) using the pHgradient-driven encapsulation protocol (L. D. Mayer et al (1989) CancerRes., 42:4734.). Briefly, a lipid composition of 90:10 DPPC:MPPC wasdissolved in chloroform and the solvent was evaporated under vacuum at45° C. using a rotavapor. The lipid film obtained after further dryingin the vacuum desiccator overnight was hydrated with 300 mM citratebuffer (pH 4.00) and the multilamellar vesicles formed were subjected toseven freeze-and-thaw cycles. The resulting suspension was extrudedthrough two polycarbonate membrane filters of pore size 0.1 μM at 50° C.using 300-400 psi pressure. The extruded liposomes were allowed to coolto room temperature and the pH was raised to 7.5-8.0 using 0.5 M Na₂CO₃solution.

The extruded liposomes were incubated at 60° C. for five minutes beforeadding pre-heated DX to the suspension. Samples were further heated for10 minutes at 60° C. with intermittent vortexing. The unentrapped drugwas removed by mini-column centrifugation using Sephadex G-50 gel.

The DX entrapped liposomes were characterized by the release of DX fromliposomes in the presence of PBS as a function of time at 37° and 40°C., as well as by the cumulative release profiles of entrapped DX fromthe liposomes at various temperatures between 25°-45° C. FIG. 7represents the release of entrapped DX from liposomes as a function oftime at 37° C. and 40° C. the liposomes were incubated at 37° and 40° C.for 30 minutes and the fluorescence intensity of the released DX wasmeasured after suitable dilutions at λex=470 nm and λem=585 nm. Thepercent release was calculated by comparing these values with values forthe total release of 6-Carboxyfluorescein (obtained by the addition ofTriton X-100 to the liposome sample). As can be seen from FIG. 7, about14% of the DX was released at 37° C. after 30 minutes of incubation,whereas 73% of the drug was released at 40° C. after 30 minutes.

As with the above studies (Examples 1-3) using CF, the cumulativerelease of DX entrapped in DPPC:MPPC 90:10 liposomes was measured byincubating the samples at various temperatures between 25°-45° C. forfive minutes and measuring the released DX fluorimetrically, as shown inFIG. 8. The results showed about 23% release of DX up to 39° C. andreached 65% release at 40° C.

EXAMPLE 5 Inclusion of PEG in Liposome Bilayers

Liposomes of the present invention were modified to render them lessrecognizable by the RES, thereby enhancing their half life in the bloodcirculation.

The surface of liposomes containing DPPC:MPPC 90:10 was modified byincorporating 5 mol % of DSPE-PEG (M.W. 2000) (FIG. 11C) in the liposomecomposition. Thus, the modified composition of these liposomes wasDPPC:MPPC:DSPE-PEG-2000 (85.935:9.545:4.520).

EXAMPLE 6 Influence of Biological Fluids on Release of Liposome Contents

Surface-modified liposomes as described in Example 5 were characterizedby studying the release profiles of CF entrapped in the aqueous interiorliposomal region in the presence of either PBS or 50% bovine serum. Thefluorescent intensity of released CF was measured at λex=470 nm andλem=520 nm, and the percent release was calculated as described inExample 3.

FIG. 9A shows the release of entrapped CF at 37° to 40° C. as a functionof time, in the presence of PBS. FIG. 9B shows the release of entrappedCF at 37° to 40° C. as a function of time, in the presence of 50% bovineserum. The surface modified liposome formulation of the presentinvention was stable at physiological temperature (37° C.) in thepresence of PBS and serum, showing 1% and 7% of CF release,respectively. However, incubation of these liposomes at 40° C. showed64% (in PBS) and 76% (in serum) release of entrapped CF after fiveminutes of incubation, and reached 77% (in PBS) and 92% (in serum) after30 minutes' incubation. Cumulative release profiles of entrapped CF fromliposomes in the presence of PBS and 50% bovine serum showed 58% and 73%release, respectively (FIG. 10). While not wishing to be held to asingle theory, the enhanced release of CF in the presence of serum couldbe attributable to the interaction of certain small molecule bloodcomponents in the serum with the liposome surface.

EXAMPLE 7 Effect of Lysolipid Concentration on Transition Enthalpy ofLipid Bilyer

Various liposomes were prepared according to the methods set forthherein employing DPPC as phospholipid and MPPC as surface active agent,i.e., lysolipid. The transition enthalpy for bilayers with varyingconcentrations of DPPC and MPPC were determined as set forth in FIG. 13.As shown, the enthalpy initially decreased but then increased abruptlyto a maximum at a 50:50 DPPC/MPPC molar ratio. Thus, this concentrationlevel produces stable gel phase bilayer and the large amount oflysolipid allows for a heightened release of active agent that ispresent within the liposome. The large amounts of lysolipid released mayalso have a local therapeutic effect and therefore enhance the efficacyof drug containing liposomes

EXAMPLE 8 Effect of Loading Doxorubicin in a Liposome Below its PhaseTransition Temperature

Doxorubicin was loaded into a lysolipid-containing temperature sensitiveliposome at 37° C., i.e., a temperature below its transitiontemperature. As a result, 80 percent of the doxorubicin loaded into theliposome, which was an improvement over the 30 to 40 percent loadingthat occurs using conventional loading techniques, namely a temperatureof 60° C., which is above the phase transition of the lipid mixture.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is describedby the following claims, with equivalents of the claims to be includedtherein.

1-65. (canceled)
 66. A liposome, comprising: an active agent and aliposome interior defined by a gel-phase bilayer membrane, and whereinthe gel-phase lipid bilayer membrane comprises: (a) a first componentwhich is one or more phospholipids selected from the group consisting ofphosphatidyl cholines, phosphatidyl glycerols, phosphatidyl inositols,phosphatidyl ethanolamines, and sphingomyelins, wherein the one or morephospholipids have two acyl groups; and (b) a second component selectedfrom: (i) one or more surface active agents selected from the groupconsisting of lysolipids, bile acids, myristoyl surfactants, palmitoylsurfactants, stearoyl surfactants, glyceryl monooleate, ceramides,PEG-ceramides, C18-ether linked lysophosphatidyl choline, polyethyleneglycol-polyethylene copolymers, fatty acids, and mixtures thereof; or(ii) an active agent; and (c) wherein the active agent, if absent fromthe gel-phase lipid bilayer membrane, is present in the liposomeinterior; and (d) wherein the amount of the second component in thegel-phase lipid bilayer membrane is sufficient to increase a firstpercentage of active agent released from the liposome at the phasetransition temperature, compared to a second percentage of active agentreleased in the absence of the second component.
 67. The liposomeaccording to claim 66, wherein the second component is the active agent.68. The liposome according to claim 66, wherein the active agent is apharmacologically active agent, a flavor agent, diagnostic agent ornutritional agent.
 69. The liposome according to claim 66, wherein theactive agent is a pharmacologically active agent selected from the groupconsisting of ceramides and platelet activating factor.
 70. The liposomeaccording to claim 66, wherein the active material is within theliposome interior.
 71. The liposome according to claim 66, wherein theone or more surface active agents are selected from the group consistingof lysolipids, bile acids, myristoyl surfactants, palmitoyl surfactants,stearoyl surfactants, glyceryl monooleate, ceramides, PEG-ceramides,C18-ether linked lysophosphatidyl choline, polyethyleneglycol-polyethylene copolymers, fatty acids, and mixtures thereof
 72. Amethod of making liposomes containing an active agent entrapped withinthe liposome interior space, comprising: (a) preparing a phospholipidfilm containing a surface active agent; (b) hydrating said phospholipidfilm with an aqueous preparation containing an active agent and thesurface active agent to produce liposomes at a temperature above thephase transition temperature of the phospholipid film, wherein theamount of surface active agent contained in the aqueous preparation issufficient to provide an equilibrating amount of surface active agent inthe interior of the liposomes; and (c) cooling the liposomes to producea liposome with a gel-phase lipid bilayer.
 73. The method according toclaim 72 wherein the liposome is present in a surrounding liquid medium,and wherein the pH of the surrounding liquid medium is greater than thepH of the interior of the liposome to facilitate loading of the activeagent.
 74. The method according to claim 72 wherein the surface activeagent is selected from the group consisting of myristoyl surfactants,palmitoyl surfactants, stearoyl surfactants, polyethyleneglycol-derivatized surfactants, glyceryl monopalmitate, glycerylmonooleate, ceramides, PEG-ceramides, polyethylene glycol-polyethylenecopolymers, C-18 ether linked lysophosphatidyl choline, and mixturesthereof.
 75. The method according to claim 72 wherein the surface activeagent is lysolipid.
 76. The method according to claim 75 wherein thelysolipid is selected from the group consisting ofmonopalmitoylphosphatidylcholine (MPPC), monolaurylphosphatidylcholine(MLPC), monomyristoylphosphatidylcholine (MMPC),monostearoylphosphatidylcholine (MSPC), and mixtures thereof.
 77. Themethod according to claim 70, wherein said phospholipid isdipalmitoylphosphatidylcholine (DPPC) and said surface active agent islysolipid which is monopalmitoylphosphatidylcholine (MPPC).
 78. Themethod of claim 72, wherein the amount of surface active agent containedin the aqueous preparation in (b) is about equal to the critical micelleconcentration of said surface active agent.
 79. The method for loadingactive agents into liposomes, comprising: (a) providing a liposomecomprising a gel-phase lipid bilayer, said lipid bilayer comprisingphospholipid, wherein said lipid bilayer is present below its phasetransition temperature; and (b) exposing the lipid bilayer to an activeagent at a temperature below the phase transition temperature of thelipid bilayer to load the liposome interior with the active agent. 80.The method according to claim 79, wherein the active agent is apharmacologically active agent, a flavor agent, diagnostic agent ornutritional agent.
 81. The method according to claim 79, wherein theactive agent is a pharmacologically active agent selected from the groupconsisting of ceramides and platelet activating factor.
 82. The methodaccording to claim 79 wherein the lipid bilayer further comprises asurface active agent.
 83. The method according to claim 82 wherein thesurface active agent is selected from the group consisting of myristoylsurfactants, palmitoyl surfactants, stearoyl surfactants, polyethyleneglycol-derivatized surfactants, glyceryl monopalmitate, glycerylmonooleate, ceramides, PEG-ceramides, polyethylene glycol-polyethylenecopolymers, C-18 ether linked lysophosphatidyl choline, and mixturesthereof.
 84. The method according to claim 82, wherein the surfaceactive agent is monostearoylphosphatidylcholine (MSPC).